Polygyny and extrapair fertilizations in eastern red-winged blackbirds (Agelaius phoeniceus)

Parentage of nestling red-winged blackbirds (Agelaius phoeniceus)from an eastern population was determinedusing DNA fingerprintingtechniques. Of 235 nestlings surveyed, 58 had fingerprints excludingthemale, but none excluded the female tending the nest. Data onpairing status during the female's fertilizable period was availablefor 232 offspring; 55 (25%of 1988 nestlings, 23% of 1989 nestlings)of those were sired through extrapair copulations. Of these55 offspring, 33 could be assigned to nearby territory holders;16 of the remaining nestlings may have been sired by nearbymales that were not captured. During both years, 44% of territorialmales had more than one female nesting simultaneously on theirterritory. The number of extrapair fertilizations gained bymales increased significandy with harem size in 1 year. Paternity(die proportion of nesdings on the territory sired by die territoryholder) showed a positive but nonsignificant increase widi haremsize in bodi years. There was no apparent cost in paternityfor males guarding two or more fertilizable females at the sametime. The broods of females that were fertilizable at die sametime anodier female was setding on die same territory tendedto have a greater proportion of extrapair fertilizations (0.37)than did die broods of odier females within harems (0.15). Establishedfertilizable females were chased significantly more by die territoryowner and by extrapair males when a new female was setding.There were no associations between a male's paternity or successat gaining extrapair fertilizations and his age or color-bandcombination. Overall, extrapair fertilizations had litde effecton die relationship between fledgling success and harem sizeand appeared to have a minimal impact on die overall intensityof sexual selection on males.     

Sequence analysis of a polymorphic Mhc class II gene in Pacific salmon

Polymorphism of the nucleotide sequences encoding 149 amino acids of linked major histocompatibility complex (Mhc) class II 131 and 132 peptides, and of the intervening intron (548–773 base pairs), was examined within and among seven Pacific salmon (Oncorhynchus) species. Levels of nucleotide diversity were higher for theB1 sequence than forB2 or the intron in comparisons both within and between species. For the codons of the peptide binding region of the BI sequence, the level of nonsynonymous nucleotide substitution (d_N) exceeded the level of synonymous substitution (d_S) by a factor of ten for within-species comparisons, and by a factor of four for between-species comparisons. The excess of d_N indicates that balancing selection maintains diversity at this salmonidMhc class II locus, as is common forMhc loci in other vertebrates. Levels of nucleotide diversity for both the exon and intron sequences were greater among than within species, and there were numerous species-specific nucleotides present in both the coding and noncoding regions. Thus, neighbor-joining analysis of both the intron and exon regions provided phylogenies in which the sequences clustered strongly by species. There was little evidence of shared ancestral (trans-species) polymorphism in the exon phylogeny, and the intron phylogeny depicted standard relationships among the Pacific salmon species. The lack of shared allelicB1 lineages in these closely related species may result from severe bottlenecks that occurred during speciation or during the ice ages that glaciated the rim of the north Pacific Ocean approximately every 100 000 years in the Pleistocene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U34692-U34720     

Ontogeny of catabolic and morphological properties of skeletal muscle of the red-winged blackbird (Agelaius phoeniceus)

Thermogenic capabilities of red-winged blackbirds improve markedly during their 10-12-day nestling period, especially between day 5 and day 8. The time course of improvements may be determined by the maturation of skeletal muscles involved in shivering thermogenesis, particularly the pectoralis muscles. To test this hypothesis, morphological and biochemical changes in pectoral and leg muscles were measured in young and adult blackbirds. Both muscles grew disproportionately relative to body mass. The pectoralis consisted entirely of fast-twitch fibers, predominantly fast oxidative glycolytic. In contrast, the gastrocnemius muscle consisted of a mixture of slow and fast fibers (predominantly fast glycolytic). Although fiber composition was constant, both cross-sectional area and density of fibers increased with age in both muscles. Catabolic capacities of the pectoralis increased significantly (approximately 7-8-fold) throughout the nestling period, most abruptly after day 3 (citrate synthase, CS) or day 4 (3-hydroxacyl-CoA-dehydrogenase, HOAD). Myofibrillar ATPase activities in the pectoralis were initially low, but increased after day 5. Further increases in CS and myofibrillar ATPase activities occurred in the pectoralis after fledging. CS and HOAD activities in the leg were much lower, but myofibrillar ATPase activities were remarkably similar in the two muscles, differing only in adults. These results are consistent with the hypothesis that the development of endothermy is dependent on the morphological and biochemical maturation of skeletal muscles important in thermogenesis.     

Plasma LH and androgen levels in the red-winged blackbird (Agelaius phoeniceus) treated with a potent GnRH analogue.

1. Low doses of GnRH-A (0.01-0.10-1.0 micrograms) given during the annual testes growth period did not clearly affect plasma LH and androgen levels 10 min following the injection. 2. The first injection of high doses of GnRH-A (2.0-10.0-20.0 micrograms) markedly increased plasma LH and androgen levels measured 10 min following the injection. The increase in plasma LH level was dose-dependent and the maximal LH level was obtained with 10.0 micrograms of GnRH-A. 3. Impairment of the LH response to GnRH-A was assessed by comparing the first and the fourteenth injection of high doses of GnRH-A. Evidences of pituitary gland desensitization are reported since plasma LH levels were reduced following the fourteenth injection in all groups. 4. Plasma androgen levels following high doses of GnRH-A were not clearly affected in red-winged blackbirds.     

Red-winged blackbirds (Agelaius phoeniceus) with higher baseline glucocorticoids also invest less in incubation and clutch mass

Glucocorticoid hormones facilitate responses to environmental challenges by mediating diverse physiological and behavioral changes, including resource mobilization and altered reproductive effort. Elevated glucocorticoids might indicate that an individual is facing high levels of environmental challenges and thus, elevated concentrations might be associated with reduced fitness (CORT-fitness hypothesis). Alternatively, the energetic demands of reproduction might be a challenge that requires elevated glucocorticoids to mobilize resources to support reproductive effort, ultimately increasing reproductive investment and fitness (CORT-adaptation hypothesis). Investigations of glucocorticoid-fitness relationships have yielded mixed results. Variation in the direction of this relationship could be caused in part by differences in the contexts in which the relationship was assessed. Incorporating context, such as life history stage, could be key to understanding the role of glucocorticoids in influencing fitness outcomes. We investigated the relationship between corticosterone and reproductive effort and success within a single life history stage: incubation of eggs. In an observational study, we measured baseline corticosterone in incubating female red-winged blackbirds (Agelaius phoeniceus), monitored incubation behavior, and determined hatching success for each nest. Incubating birds with higher baseline corticosterone concentrations had more frequent, shorter incubation bouts and spent less time overall incubating their clutches of eggs than birds with lower corticosterone concentrations. Elevated corticosterone was also associated with lower clutch mass, but neither corticosterone nor incubation effort were correlated with hatching success. Although experimental tests are needed to establish causation, these results suggest that during the incubation period, corticosterone might shift resource investment towards self-maintenance, and away from current reproductive effort.     

Songbird Genomics: Analysis of 45 kb Upstream of a Polymorphic Mhc Class II Gene in Red-Winged Blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative γ-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus)

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species. The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB301478, AB301944–AB301950, AB302087–AB302090, AB302190–AB302192, AB302843, AB302844, and AB303942–AB303945.     

Hitchhiking and recombination in birds: evidence from Mhc-linked and unlinked loci in Red-winged Blackbirds (Agelaius phoeniceus)

Hitchhiking phenomena and genetic recombination have important consequences for a variety of fields for which birds are model species, yet we know virtually nothing about naturally occurring rates of recombination or the extent of linkage disequilibrium in birds. We took advantage of a previously sequenced cosmid clone from Red-winged Blackbirds (Agelaius phoeniceus) bearing a highly polymorphic Mhc class II gene, Agph-DABI, to measure the extent of linkage disequilibrium across approximately 40 kb of genomic DNA and to determine whether non-coding nucleotide diversity was elevated as a result of physical proximity to a target of balancing selection. Application of coalescent theory predicts that the hitchhiking effect is enhanced by the larger effective population size of blackbirds compared with humans, despite the presumably higher rates of recombination in birds. We surveyed sequence polymorphism at three Mhc-linked loci occurring 1.5-40 kb away from Agph-DAB1 and found that nucleotide diversity was indistinguishable from that found at three presumably unlinked, non-coding introns (beta-actin intron 2, beta-fibrinogen intron 7 and rhodopsin intron 2). Linkage disequilibrium as measured by Lewontin's D' was found only across a few hundred base pairs within any given locus, and was not detectable among any Mhc-linked loci. Estimated rates of the per site recombination rate p derived from three different analytical methods suggest that the amounts of recombination in blackbirds are up to two orders of magnitude higher than in humans, a discrepancy that cannot be explained entirely by the higher effective population size of blackbirds relative to humans. In addition, the ratio of the number of estimated recombination events per mutation frequently exceeds 1, as in Drosophila, again much higher than estimates in humans. Although the confidence limits of the blackbird estimates themselves span an order of magnitude, these data suggest that in blackbirds the hitchhiking effect for this region is negligible and may imply that the per site per individual recombination rate is high, resembling those of Drosophila more than those of humans.     

Responses to tones of single cells in nucleus magnocellularis and nucleus angularis of the redwing blackbird (Agelaius phoeniceus)

Summary Responses to tones were recorded from single cells in the brainstem auditory nuclei n. magnocellularis and n. angularis of the redwing blackbird (Agelaius phoeniceus). Whereas 30% of the cells in angularis had rates of spontaneous activity less than 40/s, only 5% of those in magnocellularis had rates this low. Two types of response maps are found in n. angularis. Type III maps have a single excitatory area with regions of inhibition on one or both sides. Type IV maps have a region of excitation at sound levels near threshold at the best frequency of the cell; at higher levels the best frequency is inhibitory; there can be broad bands of inhibition or interleaved bands of inhibition and excitation at higher levels. Cells in magnocellularis show only Type III response maps with generally weaker inhibitory sidebands than those in angularis. Responses to excitatory tone bursts in magnocellularis are primary-like in that they show a rapid increase in discharge rate at tone onset followed by a decrease in rate of 30 to 70% over the next 50 ms. On the other hand, a number of response types are seen in angularis, including primary-like, pauser, and onset. These results for magnocellularis and angularis are compared with results from their presumed mammalian homologues, the anteroventral cochlear nucleus, and posteroventral and dorsal cochlear nuclei, respectively. Finally, the relationship between response patterns in these avian brainstem nuclei and those seen in higher auditory centers is considered.This work was supported by a grant from the National Institute of Neurological and Communicative Diseases and Stroke. J. Sinnott is a National Institutes of Health Postdoctoral Fellow. M.B. Sachs is a National Institutes of Health Research Career Development Awardee. Mrs. Phyllis Taylor worked out many of the histologic procedures and handled all of the histology for the project. Orlando Asuncion was responsible for the care of the birds used in this study. We wish to gratefully acknowledge the cooperation of the Patuxent Wildlife Center, Laurel, Maryland, and in particular Brooke Meanly.     

Habituation,Recovery and the Similarity of Song Types within Repertoires in Red-winged Blackbirds (Agelaius phoeniceus) (Aves,Emberizidae)

Male red-winged blackbirds (Agelaius phoeniceus) respond to playback of conspecific song on their territories with the song spread, a graded aggressive display in which males extend their wings to expose their red epaulets while singing. We show that the intensity of song spread display declines with repeated presentation of one song type, and recovers when song types are switched. Recovery is greater for switches between song types that are acoustically dissimilar than for switches between song types that are acoustically similar. Recovery is no different for switches between two song types taken from the repertoires of different males than for switches between song types recorded from the same male. Analysis of acoustic features also indicates that song types recorded from different males are not more dissimilar than are song types from the same male. Our results do not support the idea that repertoires of red-winged blackbirds are composed of similar song types in order to facilitate individual recognition. Rather, repertoires may be constructed of dissimilar song types, so as to help maintain the response of listeners despite habituation.     

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.     

DPB1 * BR: an Mhc class II DPB1 allele (DPB1 * 6601) of negroid origin

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X96986. The nameDPB1 ^* 6601 was officially assigned by the WHO Nomenclature Committee in May 1996. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1995), names will be assigned to new sequences as they are identified. Lists of such sequences will be published in the following WHO Nomenclature Report     

Complex regulation of class II gene expression: analysis with class II mutant cell lines

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.